This Application discloses a rapid PCR method, with particular focus on a rapid multiplex QRT-PCR method and related compositions and apparatuses.
Polymerase chain reaction (PCR) is a powerful tool in the field of molecular biology. This technique allows for replicating/amplifying trace amounts DNA fragments into quantities that can be analyzed in a meaningful way. As such this technology has been adapted to molecular biological applications like DNA sequencing, DNA fingerprinting, etc. Additionally, this method has the ability to detect specific DNA fragments in samples, whose presence may reflect a pathological state. Therefore, this method is finding new applications in the field of molecular diagnostics. Furthermore with the development of real-time quantitative PCR (QPCR), this technology has become more reliable as well as amenable to automation. Currently, this technology is used for the detection of viral and bacterial pathogens in clinical samples and for the detection of cancer cells in patients with a history of leukemias (and other cancers such as those that arise in the breast, lung, colon, esophagus and skin).
PCR in molecular diagnostics, despite its advantages, has several shortcomings. Often, this is a technique that is dependent on the technical expertise of the operator. Additionally, it is also very prone to contamination. The combination of these two above-mentioned factors results in false negative and false positive results respectively. Therefore, there is a need to incorporate internal controls along with the target of interest as well as automate the process to ensure operation within a closed system (to eliminate contamination). The incorporation of controls involves the amplification of several different sets of DNA fragments in addition to the target of interest. These controls provide information about the quality of the samples being analyzed as well as that of the assay in any given run. For technical reasons, analyzing multiple DNA targets within a sample in the same reaction tube through PCR (known as multiplexing) does not work well when the targets are not present in similar abundance at the beginning of the reaction. Historically, investigators have attempted to overcome this by limiting the primers in the PCR reaction. This approach is based on the idea that by limiting the reagents in one reaction, it stops the reaction at a point after adequate amplification has occurred for that target but before inhibition of amplification of other sequences. Though this method can increase the difference in initial abundance between the two targets that will still result in the successful amplification of both targets, it does not allow for the detection of a rare target in the milieu of a second target that is several orders of magnitude more abundant. Furthermore, when a rapid QPCR assay is called for, decreasing the primer concentration also worsens quantitation.
A further limitation of current PCR technologies is the time it takes to perform PCR diagnoses. Typical PCR reactions take hours, not minutes. As described below, decreasing the time it takes to carry out a PCR reaction is desirable for many reasons. Therefore, there is a need for an automated PCR based point of care molecular diagnostic system, especially a rapid, multiplexed (RT-)PCR assay.